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Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations

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ABSTRACT

Purpose

Antimicrobial preservatives are known to interact with proteins and potentially affect their stability in aqueous solutions. In this systematic study, the interactions of a model peptide with three commonly used preservatives, benzyl alcohol, phenol and m-cresol, were evaluated.

Methods

The impact on peptide oligomerization was studied using GC-MALS, SEC-MALS and DLS, antimicrobial efficiency of different formulations were studied using the Ph. Eur. antimicrobial efficacy test, and the molecular adsorption of preservative molecules on reversible peptide oligomers was monitored using NMR.

Results

The hydrodynamic radius and molar mass of the peptide oligomers was shown to clearly increase in the presence of m-cresol but less significantly with phenol and benzyl alcohol. The increase in size was most likely caused by peptide self-interactions becoming more attractive, leading to reversible oligomerization. On the other hand, increasing the concentration of peptide in multi-dose formulations led to reduced molecular mobility and decreased antimicrobial efficacy of all preservatives.

Conclusions

Peptide-preservative interactions not only affect peptide self-interactions, but also antimicrobial efficiency of the preservatives and are thus of significant relevance. Adsorption of preservatives on oligomeric states of peptides is proposed as a mechanism to explain this reduced antimicrobial efficacy.

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Abbreviations

\( {\overline{\mathrm{M}}}_{\mathrm{w}} \) :

Weight average molar mass

\( {\overline{\mathrm{r}}}_{{}_{\mathrm{H}}} \) :

Hydrodynamic radius

A2 :

Second virial coefficient

AET:

Antimicrobial efficacy testing

C0 :

Preservative concentration required to kill/inactivate all microbes in the absence of peptide

CFU:

Colony forming unit

CG-MALS:

Composition-gradient multi-angle light scattering

Ci :

Preservative concentration required to kill/inactivate all microbes in the presence of peptide

Dfree :

Preservative diffusion coefficient in the absence of peptide

DLS:

Dynamic light scattering

Dobs :

Preservative diffusion coefficient in the presence of peptide

DOSY:

Diffusion ordered NMR spectroscopy

Dpep :

Peptide diffusion coefficient

f:

Fraction of live microbes after incubation

fs :

Sequestered preservative fraction (in AET experiments)

Mw :

Apparent molar mass

NMR:

Nuclear magnetic resonance

pI:

Isoelectric point

PO/W :

Octanol-water partition coefficient

Ppep :

Peptide-bound preservative fraction (in NMR experiments)

SEC-MALS:

Size-exclusion chromatography, coupled with multi-angle light scattering detector

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors wish to acknowledge Claudia Bleyer and Olivier Ortschitt (F. Hoffmann – La Roche Ltd.) for their work with preservative content analyses, Dr. Arne Rufer for his work with AUC analyses, Dr. Sonoko Kanai and Michaela Grass (F. Hoffmann – La Roche Ltd.) for participating in the light scattering experiments and Jenny Train and Anita De Vivo (F. Hoffmann – La Roche Ltd.) for their assistance in manufacturing the sterile formulations for antimicrobial efficacy studies. Furthermore, Dr. Kishore Ravuri (F. Hoffmann – La Roche Ltd.) is acknowledged for his participation in experimental planning and Dr. Sulabh Patel and Tobias Werk (F. Hoffmann – La Roche Ltd.) for their input in interpreting the AET results. Finally, we would also like to acknowledge Prof. Jörg Huwyler (University of Basel, Switzerland) for reviewing this manuscript.

Author information

Authors and Affiliations

  1. Pharmaceutical Development & Supplies, Pharma Technical Development Biologics Europe (PTDE), F. Hoffmann – La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland

    P. Heljo, A. Pappenberger & H-C. Mahler

  2. Discovery Technologies, Small Molecules Research (SMR), Pharma Research and Early Development (pRED), F. Hoffmann – La Roche Ltd., Basel, Switzerland

    A. Ross

  3. Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California, USA

    I. E. Zarraga

  4. Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland

    P. Heljo

Authors
  1. P. Heljo
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  2. A. Ross
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  3. I. E. Zarraga
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  4. A. Pappenberger
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  5. H-C. Mahler
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Corresponding author

Correspondence to P. Heljo.

Electronic supplementary material

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Supplement 1

The Stejskal-Tanner type plots (22) below show the logarithm of spectral amplitudes in DOSY type spectra [ln(I/I0)], which are displayed in dependence of the square of the gradient (G2). Diffusion constants were fitted by use of the analysis module included in the NMR software package Topspin (Bruker, Fällanden, Switzerland). Ratios of diffusion constants for excipients in presence and absence of peptides (shown in the plots) were used to estimate adsorbed fractions of the excipients on the peptide or oligomers thereof according to Eqs. 4–6. The graphs in black show the measurements of 5 mg/ml preservative in the absence of peptide, and the gray ones the measurements of 5 mg/ml preservative with 10 mg/ml peptide. (GIF 6259 kb)

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Heljo, P., Ross, A., Zarraga, I.E. et al. Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations. Pharm Res 32, 3201–3212 (2015). https://doi.org/10.1007/s11095-015-1697-z

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  • DOI: https://doi.org/10.1007/s11095-015-1697-z

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